Antibody discovery programmes have become an important source of both therapeutic biomolecules and research reagents. However, the process of obtaining specific antibodies, particularly for use in therapy, remains time consuming and empirical.
The genetic basis for the structural diversity of antibodies is partially encoded in the germ line, but is also the result of stochastic genetic events, including chromosomal rearrangements, nontemplated nucleotide insertions and somatic hypermutation. The majority of this diversity is localised to the variable heavy (VH) and variable light (VL) antibody regions, and principally to the complementarity-determining regions (CDRs), which are the six-peptide loops that protrude from the variable domain framework to form the antigen-combining surface of the antibody molecule. Three CDR loops are contributed by the heavy chain (VH—CDR1, VH—CDR2 and VH—CDR3) and three by the light chain (VL—CDR1, VL—CDR2 and VL—CDR3). CDR1 and CDR2 are encoded in the germ line, and are thus relatively constrained in their diversity. VL—CDR3 is formed during the recombination of the light chain V and J genomic fragments whilst VH-CDR3 is formed by two consecutive genetic rearrangements, first between D and J and then between V and DJ. This rearrangement is additionally accompanied by the addition of non-templated nucleotides, making VH-CDR3 the source of most naturally occurring antibody diversity.
The use of combinatorial phage display single chain fragment variable (scFv) libraries for generation of therapeutic antibodies is well established and has resulted in clinically valuable reagents. ScFv libraries are made from immune or naïve B cells or as synthetic libraries where antibody VH and VL gene segments are rearranged in vitro with synthetic CDRs coding for random sequences of varying lengths. A drawback of the above method, however, is that target specific scFv binders can bind to any epitopes on the target antigen. In the case of development of affinity reagents for therapeutic applications there is a need to develop a more robust platform technology that is able to develop reagents that have therapeutic efficacy, and thus bind to the active moiety of the target antigen.